Calcitonin gene-related protein (CGRP) is a 37-amino acid neuropeptide that is expressed in a variety of cell types in both the central and peripheral nervous systems. In many tissues, CGRP-containing fibers are closely associated with blood vessels. Among the various physiological functions reported for CGRP, the most pronounced is vasodilation. CGRP is the most powerful of the vasodilator transmitters and its vasoactive effects have been demonstrated in a variety of blood vessels, including those in the cerebral, coronary, and mesenteric vasculature.
Mounting evidence suggests that CGRP is involved in the pathophysiology of migraine headache. Migraine is thought to be associated with dilation of cerebral blood vessels and activation of the trigeminovascular system. During the headache phase of a migraine, CGRP levels are elevated in the cranial venous circulation. Successful amelioration of the headache results in normalization of CGRP levels, thus implicating CGRP in the pathophysiology of this disorder. Moreover, intravenous administration of CGRP to migraineurs induces a delayed migrainous headache in some patients. These observations suggest that inhibition of CGRP-mediated vasodilation may have therapeutic utility in the treatment of migraine headaches, and including but not limited to additional indication described herein.
Aiyar, et al.(1996, J. Biol. Chem. 271: 11325–11329) disclose the gene encoding the human calcitonin receptor-like receptor (hCRLR).
McLatchie, et al. (1998, Nature 393: 333–339) disclose the gene encoding the human receptor-activity modifying proteins (hRAMP1).
Luebke, et al. (1996, Proc. Natl. Acad. Sci., USA 93: 3455–3460) disclose the gene encoding the human receptor component protein (hRCP).
The heterodimeric CGRP receptor requires co-expression of calcitonin receptor-like receptor (CRLR) and an accessory protein called receptor activity modifying protein-1, or RAMP1. Several small molecule CGRP receptor antagonists have been shown to exhibit marked species selectivity, with >100-fold higher affinities for the human CGRP receptor than for receptors from other species. CGRP activity is mediated by the Gs-coupled G-protein coupled receptor (GPCR), CRLR, which shares 55% homology with the calcitonin receptor. McLatchie et al. (id.) disclose that functional CGRP and adrenomedullin receptors are both derived from CRLR and that the phenotype is determined by co-expression with a particular RAMP. Co-expression of CRLR with RAMP1 results in CGRP receptor pharmacology, while RAMP2 or RAMP3 co-expression produces an adrenomedullin receptor. RAMPs are relatively small (148–175 amino acids) proteins containing a single predicted membrane spanning domain, a large extracellular domain, and a short cytoplasmic domain. The molecular function of RAMPs includes cell-surface targeting and may involve direct ligand binding or indirect modulation of CRLR conformation, or both.
Doods, et al. (2000, Br. J. Pharmacol. 129: 420–423) disclose that a known small-molecule antagonist of the CGRP receptor demonstrates high affinity for the human CGRP receptor, with a Ki of 14 pM. Of particular interest was the observation that this compound exhibited 200-fold lower affinity for CGRP receptors from rat, rabbit, dog, and guinea pig, although the affinity for the marmoset receptor was reported to be similar to that for human. These authors then utilized marmoset for in vivo studies to evaluate the utility of BIBN4096BS as a potential anti-migraine agent.
It is desirable to discover new drugs which antagonize the CGRP receptor for the treatment of various disorders, including but not limited to migraine, pain, menopausal hot flash, migraine prophylaxis, chronic tension type headache, cluster headache, neurogenic or chronic inflammation, gastrointestinal disorders, type 2 diabetes, as well as CGRP agonists which may be useful in the treatment of various cardiovascular disorders. To this end, it is imperative to develop a convenient animal model which expresses a CGRP receptor that mimics human CGRP pharmacological profiles, thus allowing for in vivo efficacy and receptor occupancy studies for testing of potential modulators of CGRP receptor activity, especially human CGRP activity. The present invention addresses and meets these needs by disclosing a “humanized” version of mammalian RAMP1. Co-expression of such a RAMP1 mutant with a mammalian form of CRLR results in a CGRP receptor in which small molecule CGRP receptor antagonists display potency similar to that for the human CGRP receptor. Such a mutant will be useful in both various screening assays which are known in the art, such as cell based assays, receptor binding assays and/or radioligand binding assays, as well as the generation of transgenic animals which provide for this humanized CGRP receptor activity.